Project Summary Pseudomonas aeruginosa is an opportunistic bacterial pathogen that infects a broad range of host cell types. The ability of P. aeruginosa to kill host cells is tightly regulated by several mechanisms including cell-to-cell communication and surface attachment. Metabolic pathways also appear to have a significant role in controlling the activity of host killing factors. However, the regulatory connections between central metabolism and virulence activation are not understood. What are the metabolic dynamics during P. aeruginosa activation? How do central metabolic pathways contribute to virulence induction? The work proposed here attempts to address these questions by simultaneously measuring metabolic activity and virulence states in P. aeruginosa. Metabolic activity will be measured using fluorescence lifetime imaging microscopy (FLIM), which measures the activity of the metabolite nicotinamide adenine dinucleotide (NAD(P)H), and through biochemical assays. The FLIM technique does not damage cells and does not require any chemical treatments of the cells. FLIM has been used extensively to measure metabolic activity in mammalian cells but has been used in bacteria in only a few cases. In particular, the use of FLIM to track NAD(P)H activity in P. aeruginosa has not been established. In Aim 1, our experiments will characterize the extent to which FLIM measures the relative abundance of NAD(P)H in P. aeruginosa. We will determine the extent to which molecules that are secreted by P. aeruginosa contribute to the FLIM signal and the extent to which inhibiting metabolic activity produces changes in the FLIM signal. In Aim 2, we will characterize how metabolic activity correlates with the activation of virulence. We will induce virulence in the major P. aeruginosa virulence systems and measure metabolic activity using FLIM and biochemical assays. In preliminary experiments, we observed that metabolic activity indeed correlates with the virulence state. Moving forward, we will test the converse question: does altering metabolic activity affect virulence? We will metabolically engineer P. aeruginosa to have altered NAD(P)H activity and measure potential changes in virulence. If successful, this work will establish a non-invasive procedure for real-time quantification of metabolic activity in live P. aeruginosa cells and will lay the foundation for future metabolism-virulence studies in P. aeruginosa and other pathogens. The long-term goals of this work are to develop the FLIM technique to measure metabolic dynamics in both hosts and pathogens, as our experiments enable the simultaneous collection of metabolic data from both organisms. We will extend the technique to monitor host and bacterial metabolism during acute and chronic infections in tissue and whole organism models. The results of this work will broadly impact our understanding of P. aeruginosa physiology, bacterial metabolism, and regulation of virulence by metabolism, and will potentially develop a new tool for diagnosing infection in healthcare settings.